Homogeneous detection of nucleic acid sequences is well known. Detection may include a dye, for example SYBR Green, that fluoresces in the presence of double-stranded amplification reaction product or a fluorescently labeled oligonucleotide hybridization probe. For hybridization probes, “homogeneous detection” means detection that does not require separation of bound (hybridized to target) probes from unbound probes. Among probes suitable for homogeneous detection are linear probes labeled on one end with a fluorophore and on the other end with a quencher whose absorption spectrum substantially overlaps the fluorophore's emission spectrum for FRET quenching (5′ exonuclease probes described in, for example, Livak et al. (1995) PCR Methods Appl. 4:357-362), hairpin probes labeled on one end with a fluorophore and on the other end with a quencher (molecular beacon probes described in, for example, Tyagi et al. (1996) Nature Biotechnology 14:303-308), double-stranded probes having a fluorophore on one strand and a quencher on the other strand (yin-yang probes described in, for example, Li et al. (2002) Nucl. Acids Res. 30, No. 2 e5), linear probes having a fluorophore that absorbs emission from a fluorescent dye and re-emits at a longer wavelength (probes described in, for example, United States published patent application US2002/0110450), and pairs of linear probes, one labeled with a donor fluorophore and one labeled with an acceptor fluorophore that hybridize near to one another on a target strand such that their labels interact by FRET (FRET probe pairs described in, for example, U.S. Pat. No. 6,140,054). Detection methods include methods for detecting nucleic acid sequences in single-stranded targets, double-stranded targets, or both.
Nucleic acid target sequences suitable for probing can in some instances be obtained directly by isolation and purification of nucleic acid in a sample. In other instances nucleic acid amplification is required. Amplification methods for use with homogeneous detection include the polymerase chain reaction (PCR), including symmetric PCR, asymmetric PCR and LATE-PCR, any of which can be combined with reverse transcription for amplifying RNA sequences, NASBA, SDA, and rolling circle amplification. Amplification-detection methods may rely on fluorescence due to probe hybridization, or they may rely on digestion of hybridized probes during amplification, for example, the 5′ nuclease amplification-detection method. If a sample contains or is amplified to contain, double-stranded target, for example, the amplification product of a symmetric PCR reaction, but single-stranded target is desired, separation of plus and minus strands can be accomplished by known methods, for example, by labeling one primer with biotin and separating the biotin-containing product strands from the other strands by capture onto an avidin-containing surface, which is then washed.
Certain fluorescent probes useful for homogeneous detection contain a fluorophore-labeled strand that emits a detectable signal when it hybridizes to its target sequence in a sample. For example, a molecular beacon probe is single-stranded and emits a detectable fluorescent signal upon hybridization. A ResonSense® probe is also single stranded and signals only when hybridized provided that the sample contains a dye, generally a SYBR dye, which stimulates hybridized probes by FRET when the dye is stimulated. Yin-yang probes are quenched double-stranded probes that include a fluorophore-labeled strand that emits a detectable signal it hybridizes to its target. FRET probe pairs, on the other hand, are probe pairs that emit a detectable fluorescent signal when both probes of the pair hybridize to their target sequences. Some amplification assays, notably the 5′ nuclease assay, include signal generation caused by probe cutting to generate fluorescent probe fragments rather than simply probe hybridization.
Certain probes that generate a signal upon hybridization can be constructed so as to be “allele-specific,” that is, to hybridize only to perfectly complementary target sequences, or to be mismatch-tolerant, that is, to hybridize to target sequences that either are perfectly complementary to the probe sequence or are generally complementary but contain one or more mismatches. Allele-specific molecular beacon probes have relatively short probe sequences, generally single-stranded loops not more than 25 nucleotides long with hairpin stems 4-6 nucleotides long, and are useful to detect, for example, single-nucleotide polymorphisms. Marras et al. (1999) Genetic Analysis: Biomolecular Engineering 14: 151-156, discloses a real-time symmetric PCR assay that includes in the reaction mixture four molecular beacons having 16-nucleotide long probe sequences and 5-nucleotide stems, wherein each probe is a different color, that is, includes a fluorophore that is detectably distinguishable by its emission wavelength, and a probe sequence differing from the others by a single nucleotide. The sample is analyzed after each PCR cycle to detect which color arises and thereby to identify which of four possible target sequences perfectly complementary to one of the probes is present in a sample. Mismatch-tolerant molecular beacon probes have longer probe sequences, generally single-stranded loops of up to 50 or even 60 nucleotides with hairpin stems maintained at 4-7 nucleotides. Tyagi et al. European Patent No. 1230387 discloses a symmetric PCR amplification and homogeneous detection assay using a set of four differently colored mismatch-tolerant molecular beacon probes having different probe sequences 40-45 nucleotides long and stems 5-7 nucleotides long, to hybridize competitively to, and thereby interrogate, a 42-nucleotide long hypervariable sequence of mycobacterial 16S rRNA genes to determine which of eight mycobacterial species is present in a sample. The sample is analyzed by determining a ratio of fluorophore intensities at one or more temperatures to identify the species that is present. El-Hajj et al (2009) J. Clin. Microbiology 47:1190-1198, discloses a LATE-PCR amplification and homogeneous detection assay similarly using four differently colored mismatch-tolerant molecular beacon probes having different probe sequences 36-39 nucleotides long and stems 5 nucleotides long to hybridize competitively to, and thereby interrogate, a 39-nucleotide long hypervariable sequence of mycobacterial 16S rRNA genes to determine which of twenty-seven mycobacterial species is present in a sample. Each of the four probes is a “consensus probe,” that is, it has a single-stranded loop complementary to multiple species but perfectly complementary to none of them. Genomic DNA from some 27 different species were separately amplified, the Tm of each probe was determined by post-amplification melt analysis, and data was tabulated. To analyze a sample containing an unknown species, the sample was amplified and analyzed as above. The Tm's of all four probes were compared to the tabulated results to identify the species present in the sample.
Multiple probes, both mismatch-tolerant and allele-specific, have been used to interrogate multiple target sequences as well as target sequences longer than a single allele-specific probe. Allele-specific molecular beacon probes have been utilized to interrogate sequences longer than one probe sequence under either of two approaches. Piatek et al. (1998) Nature Biotechnology 16:359-363, discloses performing parallel, real-time, symmetric PCR amplification assays, each containing one of five, fluorescein-labeled, allele-specific molecular beacons which together span an 81-nucleotide long sequence of one strand of the rpoB gene core region of M. tuberculosis in overlapping fashion. Analysis was detection of probe fluorescence intensities after each PCR cycle. Failure of any one of the probes to hybridize to PCR-amplified target sequence (“amplicon”) and emit its fluorescent signal was taken as an indication of drug resistance. El-Hajj et al. (2001) J. Clin. Microbiology 39:4131-4137, discloses performing a single, multiplex, real-time, symmetric PCR assay containing five differently colored, allele-specific molecular beacons, three complementary to one amplicon strand and two complementary to the other amplicon strand, which together span an 81-nucleotide long region of the rpoB gene core region of M. tuberculosis in overlapping fashion. Here again, probe fluorescence intensities were obtained, and failure of any one of the probes to hybridize and signal was taken as an indication of drug resistance. Wittwer et al. U.S. Pat. No. 6,140,054 discloses a multiplex symmetric PCR assay for detecting single and double base-pair mismatches in two sequences (C282Y and H63D sites) of the human HFE gene using a primer pair for each site, a FRET probe pair for each site, and rapid thermal cycling. Each probe pair includes a mismatch-tolerant fluorescein donor probe 20-30 nucleotides in length, positioned to hybridize to target sites of possible variations, and a Cy5 acceptor probe 35-45 nucleotides long, called the “anchor” probe, because it remains hybridized as its companion fluorescein probe melts off the target sequence at a melting temperature dependent on its degree of complementarity. The probe pair for one site, the C282Y site has a lower Tm range for wild type and mutant targets than does the probe pair for the H63D. Each probe pair has a higher melting Tm against its mutant target than against its wild type target As described by Witter, the melting temperature of at least one of the probes, typically the acceptor probe, is above the annealing temperature of both of the primers used in a symmetric PCR amplification, and the reaction kinetics are followed in real-time. Following amplification, a sample is analyzed by determining the Tm's of both probe pairs from the emissions of the acceptor (Cy5) probes. A target sequence having a single-nucleotide mismatch to its fluorescein-labeled donor probe, that is, a wild-type sequence, causes the donor probe to melt at a lower temperature, thereby lowering the melting temperature by about 5° C., revealing the presence of a mismatch. The genotype of a genome is established as either homozygous or heterozygous based on whether a signal is observed at one or two specific temperatures whose positions are anticipated in advance. Heterozygous genomes have equal concentrations of two possible alleles.
Analysis of nucleic acid sequences using multiple probes for long target sequences, whether a long single target sequence or multiple target sequences, by the foregoing methods is limited by the amount of information that can be obtained. In FRET-probe analysis, for every donor probe whose melting behavior is detected, there is a corresponding acceptor probe of high Tm that serves simply as an “anchor” and does not interrogate the target in a detectable fashion. Methods using molecular beacons, whether allele-specific or mismatch-tolerant, are limited by the number of fluorophore colors that can be distinguished in a single reaction mixture (maximally seven or eight for some detection instruments but only four colors for other instruments), and certain molecular-beacon methods are limited to relatively short target sequences. U.S. Pat. No. 7,385,043 discloses an assay intended to overcome the color limitation. It discloses a screening assay for one among as many as fifty or even seventy possible targets by having a probe specific to each target, specifically an allele-discriminating molecular beacon probe, subdividing each probe into multiple parts, and labeling each part with a different fluorophore, to create a multi-color code identifying each probe. Assays utilizing this approach are complicated and, thus, expensive, because the probes must have multiple fluorophores.
Sepsis exemplifies the need to analyze long nucleic acid target sequences. Analysis of sepsis is further complicated by the need to differentiate among numerous bacterial species, any of which could be the cause of infection. There is a need for single-tube screening assays for pathogenic infections such as sepsis, particularly assays that can be performed in laboratories other than high-complexity CLIA laboratories, that is, point-of-care diagnostic laboratories located at or near the site of patient care.